Signalingfrommembranestotheactincytoskeletonunderpinsamyriadofprocesses,suchascellmigration andvesicletrafficking,anditsdysregulationleadstovariousdiseases,includingcancerandneurologicaldis- orders.TheWAVERegulatoryComplex(WRC)of~400kDaplaysacentralroleinlinkingmembranesignals totheactincytoskeletonthroughoutbiology.TheWRCaloneisautoinhibited,butalargevarietyofmembrane ligandscanactivatethecomplexthroughdirectinteractions,which,inturn,stimulatestheArp2/3complexto polymerizeactin.TheseligandsincludetheGTPasesRac1andArf1,andover100differentmembranepro- teinspreviouslyidentifiedbytheapplicant.DespitethislonglistofWRCligandsandtheirbroadbiologicalfunc- tions,itremainsunknownhowthesemembranemolecules,individuallyorcooperatively,controlWRC?sactivity andtherebyactinassembly.Onemajorchallengeistobiochemicallyreconstitutethemultivalent,weakinterac- tionsoftheWRCwithitsligands,especiallywithitscanonicalactivatorsRac1andArf1.Preliminarydata demonstratethatitisnowpossibletoreconstitutestableWRC/ligandcomplexesbyusingrecombinantmateri- alandbydifferenttetheringstrategies.Theuniqueaccesstosuchmaterialwillenabledeterminationofthebi- ochemicalandstructuralmechanismsmediatingWRCactivation.Preliminarydatasuggestthatactivationof theWRCrequiressimultaneousbindingoftwoRac1moleculesattwodistinctsitesandthattheactivationis enhancedbyathirdinteractionwithArf1.Othermembraneligandsmayfurthermodulatetheactivationbyin- teractionsatadditional,distinctsites.MylabwilltargetthreemajoraspectsofWRCactivationbybiochemical andstructuralapproaches.Project1willdeterminethestructureoftheWRCsimultaneouslyboundtotwo Rac1molecules.ThiswillrevealthefirstactivatedstructureoftheWRCandwillprovideamechanisticframe- workforunderstandingWRCactivation.Project2willcombinebiochemicalandstructuralapproachestodefine howtheWRCisactivatedbyArf1andhowArf1andRac1actcooperativelytooptimizeactivation.Thiswillre- vealnewmechanismsofWRCactivationandopennewavenuesforunderstandingactinregulationinmany processescontrolledbytheArffamilyofGTPases.Project3willdiscovernovelinteractionmechanismsofthe WRCwiththreenewlyidentifiedmembraneligands,includingtheclaudin-likereceptorHPO-30,theneuronal receptorRetrolinkin,andthekainatefamilyofglutamatereceptors.Thiswillrevealnewmodesofactinregula- tionmediatedbymembranereceptorsandtheWRC.Together,bygeneratingpreviouslyunattainablematerial andbycloselyintegratingquantitativebiochemistryandstructuralapproaches,successfulcompletionofthis workwillprovideacomprehensive,unifyingframeworkforunderstandingWRCactivation,knowledgethatwill bebroadlyapplicabletomanydifferentcellularsystemsandbiologicalprocesseswidelyregulatedbythissig- nalinghubanditsmanyligands.Ourworkwillalsoprovidenewstructuraltargetsfortherapeuticsandwillhave abroadimpactinareasrangingfromcellbiologyandimmunologytoneuroscienceanddevelopmentalbiology.